Mutant KLF protein, and method for producing induced pluripotent stem cells

Abstract

There is provided a mutant KLF protein that can induce reprogramming of a somatic cell at a higher efficiency than a KLF protein having a natural amino acid sequence. There is also provided a method for efficiently producing an iPS cell by using the mutant KLF protein. There is provided a mutant KLF protein having an amino acid substitution, or a peptide fragment thereof containing the amino acid substitution.

The invention claimed is: 
     
         1 . A mutant Krüppel-like factor (KLF) protein comprising an amino acid substitution, wherein
 the amino acid substitution is a substitution of any of the following: 
 (a) serine at position 349 and/or leucine at position 356 in the amino acid sequence set forth in SEQ ID NO: 1; 
 (b) serine at position 342 and/or leucine at position 349 in the amino acid sequence set forth in SEQ ID NO: 3; 
 (c) serine at position 500 and/or leucine at position 507 in the amino acid sequence set forth in SEQ ID NO: 5; or 
 (d) serine at position 443 and/or leucine at position 450 in the amino acid sequence represented by SEQ ID NO: 7. 
 
     
     
         2 . The mutant KLF protein according to  claim 1 , wherein
 the substitution of (a) is S349A, and/or L356A, L356N, L356D, L356C, L356E, L356G, L356K, L356M, L356S, or L356T,   the substitution of (b) is S342A, and/or L349A, L349N, L349D, L349C, L349E, L349G, L349K, L349M, L349S, or L349T,   the substitution of (c) is S500A, and/or L507A, L507N, L507D, L507C, L507E, L507G, L507K, L507M, L507S, or L507T, or   the substitution of (d) is S443A, and/or L450A, L450N, L450D, L450C, L450E, L450G, L450K, L450M, L450S, or L450T.   
     
     
         3 . A nucleic acid encoding the mutant KLF protein according to  claim 1 . 
     
     
         4 . A gene expression vector comprising the nucleic acid according to  claim 3 , in an expressible state. 
     
     
         5 . An induced pluripotent stem cell (iPS cell) inducer comprising the nucleic acid according to  claim 3 . 
     
     
         6 . A direct reprogramming agent comprising the nucleic acid according to  claim 3 . 
     
     
         7 . A cancer therapeutic agent comprising the nucleic acid according to  claim 3 . 
     
     
         8 . An induced pluripotent stem cell (iPS cell) inducer comprising the gene expression vector according to  claim 4 . 
     
     
         9 . A direct reprogramming agent comprising the gene expression vector according to  claim 4 . 
     
     
         10 . A cancer therapeutic agent comprising the gene expression vector according to  claim 4 . 
     
     
         11 . An induced pluripotent stem cell (iPS cell) inducer comprising the mutant KLF protein according to  claim 1 . 
     
     
         12 . The iPS cell inducer according to  claim 11 , further comprising (i) and/or (ii):
 (i) any of an OCT3/4 protein, a nucleic acid encoding the protein, or a gene expression vector comprising the nucleic acid in an expressible state;   (ii) any of a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein or a SOX17 protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state.   
     
     
         13 . The iPS cell inducer according to  claim 12 , further comprising
 (iii) any of a C-MYC protein, a T58A mutant of the C-MYC protein, an N-MYC protein or a L-MYC protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state.   
     
     
         14 . A direct reprogramming agent comprising a mutant KLF protein according to  claim 1 . 
     
     
         15 . A method for producing an iPS cell, comprising introducing an iPS cell inducer comprising the following (1) to (3), into a somatic cell:
 (1) a mutant KLF protein according to  claim 1 ,   (2) any of an OCT3/4 protein, a nucleic acid encoding the protein, or a gene expression vector comprising the nucleic acid in an expressible state, and   (3) any of a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein or a SOX17 protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state; and   cultivating the somatic cell after the introduction step in the presence of one or more of a basic fibroblast growth factor, a TGF-β1 protein, a BMP protein, a Wnt3 protein, a GSK3β inhibitor, a Wnt inhibitor, retinoic acid, ascorbic acid, and a ROCK inhibitor.   
     
     
         16 . The production method according to  claim 15 , further comprising a selection step of selecting an iPS cell induced in the cultivation step. 
     
     
         17 . The production method according to  claim 15 , wherein the somatic cell is human-derived. 
     
     
         18 . A method for producing an iPS cell, comprising introducing an iPS cell inducer comprising the following (1) to (4), into a somatic cell:
 (1) a mutant KLF protein according to  claim 1 ,   (2) any of an OCT3/4 protein, a nucleic acid encoding the protein, or a gene expression vector comprising the nucleic acid in an expressible state,   (3) any of a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein or a SOX17 protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state, and   (4) any of a C-MYC protein, an N-MYC protein, an L-MYC protein, or a T58A mutant protein of the C-MYC protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state; and   cultivating the somatic cell after the introduction step.   
     
     
         19 . A cancer therapeutic agent comprising, the mutant KLF protein according to  claim 1 .